The genome sequence of rosebay willowherb Chamaenerion angustifolium (L.) Scop., 1771 (syn. Epilobium angustifolium L., 1753) (Onagraceae)

We present a genome assembly from an individual Chamaenerion angustifolium (fireweed; Tracheophyta; Magnoliopsida; Myrtales; Onagraceae). The genome sequence is 655.9 megabases in span. Most of the assembly is scaffolded into 18 chromosomal pseudomolecules. The mitochondrial and plastid genome assemblies have lengths of 495.18 kilobases and 160.41 kilobases in length, respectively.


Background
This plant species is known under a variety of names.In the UK and Ireland, it is commonly referred to as rosebay willowherb (as its leaves resemble those of some willows) and sometimes locally as bombweed, reflecting its ability to rapidly colonise bomb craters (e.g. after the blitz in the Second World War, Hamilton, 2022).More widely, especially in USA, it is known as fireweed as it is a frequent colonizer of the bare ground following forest fires.
The species is a perennial herb, spreading rapidly by underground stolons, and it produces large numbers of hairy wind-dispersed seeds (e.g., some studies estimate a single plant can produce around 80,000 seeds per year, Broderick, 1990), enabling the growth of large, dense stands of tall leafy (up to 2.5 m) stems, topped with spikes of fuchsia-pink flowers (Figure 1) that are attractive to a large range of insect pollinators, including honeybees.It has a circumpolar distribution, ranging from the arctic/subarctic to cool-temperate regions of North America and Eurasia, to as far south as Mexico, North Africa, the Himalayas, Myanmar and southern China.It is now common and widespread across the UK and Ireland, despite being reported to be rare in the 18th and 19th centuries, when it grew on undisturbed wet, gravelly soils.Its rise in abundance is considered, in part, to reflect its capacity to rapidly colonize and spread in response to changes of land management, an increase in human disturbance and the intensification of (controlled) burning of heathland and forests.
The genus placement of this species has been contentious as it has moved between Chamaenerion Ség., Pyrogennema Lunell, Chamerion (Raf.)Holub.and Epilobium L. by various authors.Chamaenerion was originally used for all willowherbs (Tournefort, 1700), but Linnaeus preferred the name Epilobium, placing Chamaenerion into synonymy.Baum et al. (1994) found genetic evidence in support of the acceptance of Chamerion, which was confirmed by Levin et al. (2004), who found the species as sister to the other species of Epilobium.There are also some morphological differences such as the alternate leaves, slightly zygomorphic flowers, entire petals and equal length of its eight stamens (Figure 1C, Wagner et al., 2007), but these do not separate it from all other Epilobium species, especially when the deeply embedded former genus Zauschneria is included.These also have zygomorphic flowers and frequently alternate leaves.There has also been widespread confusion on the usage of Chamerion versus the earlier name Chamaenerion, as the latter was originally believed to be illegitimate (Sennikov, 2011).Here we treat this species as Chamaenerion angustifolium as NCBI recognise it as belonging to a separate genus from Epilobium (although Epilobium angustifolium is listed as a homotypic synonym) and it is the name under which all the genomic data generated here are stored.We note that the name Epilobium refers to the position of the petals above the ovary (from Greek epi, upon, and lobos, lobe), while Chamaenerion is derived from Greek chamai, low, and nerion, oleander.The species name 'angustifolium' refers to the narrow leaves of this species.
The species is reported to be used by humans in numerous ways.For example, the pith of the stems can be eaten, and it is considered to be a good additive to soups and stews.It has a flavour of sweet cucumber with a peppery aftertaste (Kirtley, 2024).Dry stems can be used as twine or to light fires while extracts are used in some creams, shampoos and other cosmetic products, especially for acne (Adamczak et al., 2019).It is also widely used in traditional medicine as it is reported to have anti-cancer, anti-bacterial, anti-inflammatory, and anti-aging properties due to the presence of a diversity of polyphenols and secondary metabolites (reviewed in Prasad et al., 2018).
Here we present the first high-quality genome of C. angustifolium.Its genome will not only provide an important baseline resource for studying the evolution of autopolyploids in this model species, but also for helping to dissect the biochemical pathways that lead to the production of over 250 metabolites that may be bioactive and explain the widespread use of this species in traditional medicine (Adamczak et al., 2019;Kadam et al., 2018).

Genome sequence report
The genome was sequenced from a specimen of Chamaenerion angustifolium (Figure 1) collected from the Royal Botanic Gardens Kew, Richmond, Surrey, UK (51.48, -0.30).Using flow cytometry, the genome size (1C-value) was estimated to be 0.85 pg, equivalent to 840 Mb.A total of 40-fold coverage in Pacific Biosciences single-molecule HiFi long reads and 64-fold coverage in 10X Genomics read clouds was generated.Primary assembly contigs were scaffolded with chromosome conformation Hi-C data.Manual assembly curation corrected 26 missing joins or mis-joins and removed 2 haplotypic duplications, reducing the assembly length by 0.48%, and increasing the scaffold number by 14.68% and the scaffold N50 by 68.75%.
The final assembly has a total length of 655.9 Mb in 123 sequence scaffolds with a scaffold N50 of 36.6 Mb (Table 1).The snail plot in Figure 2 provides a summary of the assembly statistics, while the distribution of assembly scaffolds on GC proportion and coverage is shown in Figure 3.The cumulative assembly plot in Figure 4 shows curves for subsets of scaffolds assigned to different phyla.Most (96.59%) of the assembly sequence was assigned to 18 chromosomal-level scaffolds.Chromosome-scale scaffolds confirmed by the Hi-C data are named in order of size (Figure 5; Table 2).While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited.The mitochondrial and plastid genomes were also assembled and can be found as contigs within the multifasta file of the genome submission.
The genome size was estimated by flow cytometry using the fluorochrome propidium iodide and following the 'one-step' method as outlined in Pellicer et al. (2021).Specifically for this species, the General Purpose Buffer (GPB) supplemented with 3% PVP and 0.08% (v/v) beta-mercaptoethanol was used for isolation of nuclei (Loureiro et al., 2007), and the internal calibration standard was Petroselinum crispum 'Champion Moss Curled' with an assumed 1C-value of 2,200 Mb (Obermayer et al., 2002).The RNA concentration was assessed using a Nanodrop spectrophotometer and a Qubit Fluorometer using the Qubit RNA Broad-Range Assay kit.Analysis of the integrity of the RNA was done using the Agilent RNA 6000 Pico Kit and Eukaryotic Total RNA assay.
Protocols developed by the WSI Tree of Life core laboratory are publicly available on protocols.io(Denton et al., 2023).

Sequencing
Pacific Biosciences HiFi circular consensus and 10X Genomics read cloud DNA sequencing libraries were constructed according to the manufacturers' instructions.Poly(A) RNA-Seq libraries were constructed using the NEB Ultra II RNA Library Prep kit.DNA and RNA sequencing was performed by the Scientific Operations core at the WSI on Pacific Biosciences SEQUEL II (HiFi), Illumina HiSeq 4000 (RNA-Seq) and Illumina NovaSeq 6000 (10X) instruments.Hi-C data were also generated from leaf tissue of drChaAngu1 using the Arima2 kit and sequenced on the Illumina NovaSeq 6000 instrument.

Genome assembly, curation and evaluation
Assembly was carried out with Hifiasm (Cheng et al., 2021) and haplotypic duplication was identified and removed with purge_dups (Guan et al., 2020).One round of polishing was performed by aligning 10X Genomics read data to the assembly with Long Ranger ALIGN, calling variants with FreeBayes    et al., 2019).The assembly was checked for contamination and corrected using the gEVAL system (Chow et al., 2016) as described previously (Howe et al., 2021).Manual curation was performed using gEVAL, HiGlass (Kerpedjiev et al., 2018) and PretextView (Harry, 2022).The mitochondrial and chloroplast genomes were assembled using MBG (Rautiainen & Marschall, 2021) from PacBio HiFi reads mapping to related genomes.A representative circular sequence was selected for each from the graph based on read coverage.
Table 3 contains a list of relevant software tool versions and sources.

Wellcome Sanger Institute -Legal and Governance
The materials that have contributed to this genome note have been supplied by a Darwin Tree of Life Partner.The submission of materials by a Darwin Tree of Life Partner is subject to the 'Darwin Tree of Life Project Sampling Code of Practice', which can be found in full on the Darwin Tree of Life website here.By agreeing with and signing up to the Sampling Code of Practice, the Darwin Tree of Life Partner agrees they will meet the legal and ethical requirements and standards set out within this document in respect of all samples acquired for, and supplied to, the Darwin Tree of Life Project.
Further, the Wellcome Sanger Institute employs a process whereby due diligence is carried out proportionate to the nature of the materials themselves, and the circumstances under which they have been/are to be collected and provided for use.
The purpose of this is to address and mitigate any potential legal and/or ethical implications of receipt and use of the materials as part of the research project, and to ensure that in doing so we align with best practice wherever possible.The overarching areas of consideration are: • Ethical review of provenance and sourcing of the material

Samuli Lehtonen
Biodiversity Unit, University of Turku, Turku, Finland The article presents a genome assembly of Chamaenerion angustifolium (Epilobium angustifolium).The rationale for creating the genome assembly is explained and the work is clearly justified.The protocols applied are documented with sufficient details and are appropriate for the purpose.The datasets are clearly presented and fully accessible.
Apparently the sequenced specimen is presumed to be diploid on the basis that so far all the chromosome counts for Britain have been diploid for this species, even if variation in chromosome counts have been reported elsewhere.Perhaps this could be specifically mentioned?
The paper states that "While not fully phased, the assembly deposited is of one haplotype.Contigs corresponding to the second haplotype have also been deposited."This implies that at least some haplotype phasing has been conducted, although it is not explained in the methods.This part could be clarified.

Anze Svara
Cornell University, Ithaca, New York, USA This is a novel and valuable resource for plant research community that will enable future genomic studies of Chamaenerion species.Below are my specific remarks to the manuscript: Specific remarks Please, include a Jellyfish plot on size estimation and heterozygosity, to validate flow-cytometry results.
Is there a reason why repeat and gene annotation has not been performed?Background I suggest mentioning the plant species name in the first paragraph.1.
Second paragraph: Please, break the first long sentence into multiple sentences.

2.
"There are also some morphological differences such as the alternate…" Differences between what?Also, break this sentence up, as it is fairly long.Do the same for similar long sentences throughout the manuscript.

3.
"especially when the deeply embedded former genus Zauschneria is included".What is "embedded" referring to?Try to be more specific.

4.
"These" and "alternate leaves" What are these two fphrases referring to?Be more specific.5.
"especially for acne" Please, specify what does it do to acne.6.
Could you please explain how k-mer completeness has been calculated?7.
Genome sequence report "While not fully phased, the assembly deposited is of one haplotype."From this section, it is unclear how phasing was done and why the chromosomes have not been phased entirely.
Can you elaborate on this in the text?Also, does this mean that haplotype 1 contains additional sequences compared to haplome 2? How were falsely duplicated regions filtered? 1.
"The mitochondrial and plastid genomes were also…" Add specifications of the plastid genomes. 2.
"The estimated Quality Value (QV) …" I suggest reporting data for haplome 2 as well, i.e. each haplome individually.What is the reason for the relatively low QV? 3.
Methods "The genome was analysed within the BlobToolKit environment" What specifically has been analyzed? 1.

Parimalan Rangan
1 ICAR-National Bureau of Plant Genetic Resources, New Delhi, Delhi, India 2 QAAFI, The University of Queensland (Ringgold ID: 1974), Saint Lucia, Queensland, Australia Following are the inputs that the authors may consider adding to improve their manuscript: 1.The authors may add a summary statistics of genome assembly in a table comparing the improvement for addition of each method.For example, they used HiFi for contig assembly, then used 10x, and finally scaffolded using HiC data.So each additional information from a different platform, how much the assembly was improved.This will give readers an overview on what methods they can choose.
2. Additionally, they can perform BUSCO analysis with Viridiplantae or the nearest taxonomical order to understand the accuracy level.
3. The quality of the genome assembly can be assessed using LAI tools.The authors may look into it.4. Structural annotation using appropriate tools might help them to identify the genes present in the genome.

Is the rationale for creating the dataset(s) clearly described? Yes
Are the protocols appropriate and is the work technically sound?Yes Are sufficient details of methods and materials provided to allow replication by others?Yes Are the datasets clearly presented in a useable and accessible format?Yes Competing Interests: No competing interests were disclosed.
Reviewer Expertise: Genomics, transcriptomics, molecular biology, I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.

Figure 2 .
Figure 2. Genome assembly of Chamaenerion angustifolium, drChaAngu1.1:metrics.The BlobToolKit snail plot shows N50 metrics and BUSCO gene completeness.The main plot is divided into 1,000 size-ordered bins around the circumference with each bin representing 0.1% of the 656,518,074 bp assembly.The distribution of scaffold lengths is shown in dark grey with the plot radius scaled to the longest scaffold present in the assembly (46,612,786 bp, shown in red).Orange and pale-orange arcs show the N50 and N90 scaffold lengths (36,550,787 and 24,316,059 bp), respectively.The pale grey spiral shows the cumulative scaffold count on a log scale with white scale lines showing successive orders of magnitude.The blue and pale-blue area around the outside of the plot shows the distribution of GC, AT and N percentages in the same bins as the inner plot.A summary of complete, fragmented, duplicated and missing BUSCO genes in the eudicots_odb10 set is shown in the top right.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAMPFE01/dataset/CAMPFE01/snail.
Figure 3. Genome assembly of Chamaenerion angustifolium, drChaAngu1.1:BlobToolKit GC-coverage plot.Scaffolds are coloured by phylum.Circles are sized in proportion to scaffold length.Histograms show the distribution of scaffold length sum along each axis.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAMPFE01/dataset/CAMPFE01/blob.

Figure 4 .
Figure 4. Genome assembly of Chamaenerion angustifolium, drChaAngu1.1:BlobToolKit cumulative sequence plot.The grey line shows cumulative length for all scaffolds.Coloured lines show cumulative lengths of scaffolds assigned to each phylum using the buscogenes taxrule.An interactive version of this figure is available at https://blobtoolkit.genomehubs.org/view/CAMPFE01/dataset/CAMPFE01/cumulative.

Figure 5 .
Figure 5. Genome assembly of Chamaenerion angustifolium, drChaAngu1.1:Hi-C contact map of the drChaAngu1.1 assembly, visualised using HiGlass.Chromosomes are shown in order of size from left to right and top to bottom.An interactive version of this figure may be viewed at https://genome-note-higlass.tol.sanger.ac.uk/l/?d=GpFNn0GfQgScVp3ROZBHTA.

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the rationale for creating the dataset(s) clearly described? Yes Are the protocols appropriate and is the work technically sound? Yes Are sufficient details of methods and materials provided to allow replication by others? Yes Are the datasets clearly presented in a useable and accessible format? Yes Competing Interests:
No competing interests were disclosed.

have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard.
This is an open access peer review report distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.